Stimulation of trout CYP1A gene expression in mouse HEPA-1 cells by 3-methylcholanthrene

Abstract

Trout CYP1A-CAT expression construct was generated by cloning ∼3.5 Kb 5′ flanking DNA of trout liver CYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrocarbon receptor 1 were transfected with trout CYP1JA-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if 5′ flanking DNA of trout CYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected trout CYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogenous cyp1a1 activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells transfected with trout CYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that trout CYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus trout CYP 1A-CAT could serve as a good model to study the mechanism of regulation of CYP1A1 gene expression.